Both the commitment of progenitors to the ?? lineage and specification of their effector fates occurs during development in the thymus; however, our understanding of the developmental cues controlling these fate decisions remains incomplete. Accumulating evidence suggests that both ?? lineage commitment and effector fate are influenced by differences in T cell receptor (TCR) signal strength. The differences in TCR signal strength influence fate by inducing Id3, an antagonist of E protein DNA binding. While E proteins clearly play a central role, their influence on developmental outcomes is almost certainly modulated by additional transcription factors and the extracellular signals that control their expression and function. Accordingly, these fate decisions are too complex to be understood by focusing on one gene or pathway and so require the comprehensive, network-based approach pioneered by Murre. The overall goal of this program is to determine how ?? TCR signals of varying intensities are generated and understand the role that the resultant alterations in E protein activity and cooperating DNA-binding proteins play in influencing lineage and ?? effector fate. Gaining a comprehensive understanding of such a multifaceted developmental process is beyond the scope of any individual laboratory, as it requires facility with numerous experimental approaches to manipulate fate-determining cues and assess their effect on fate, both functionally and molecularly, in vitro and in vivo. The investigators comprising this program possess the necessary distinct, yet complementary, skills to do so. The Wiest lab (Project 1) has generated in vivo and in vitro models in which developmental fates can be manipulated by altering TCR signal intensity. Dr. Zuniga-Pflucker (Project 3) has established elegant in vitro and in vivo systems in which Notch and cytokine input can be manipulated to assess the impact on effector fate. Drs. Zhuang (Project 2) and Murre (Project 4) are experts in the genetic and molecular analysis of E proteins and their Id family antagonists. Finally, Dr. Murre (Genomics Core) will utilize his novel bioinformatic approach to assist all projects in molecularly defining critical milestones in ?? development by assembling global regulatory networks assembled around E protein targets. Collectively, these efforts promise to move the field forward by comprehensively defining the processes controlling ?? lineage commitment and effector fate from extracellular signals to the network of targets in the nucleus. RELEVANCE: ?? T cells are increasingly understood to play critical roles in immune responses to pathogens and tumors that are unique and thus not overlapping with those of ???lineage T cells. Therefore, a greater understanding of how the development of ?? T cells if controlled at the molecular level may enable the manipulation of their production or function for therapeutic benefit. Project 1: Influence of ligand on specification of gamma/delta fate and function Project Leader (PL): Wiest, D DESCRIPTION (provided by applicant): The goal of this proposal is to understand the molecular processes controlling ?? lineage commitment and specification of effector fate. Both events occur during development in the thymus; however, our understanding of the developmental cues controlling these fate decisions remains incomplete. Accumulating evidence suggests that both ?? lineage commitment and effector fate are influenced by differences in T cell receptor (TCR) signal strength. The differences in TCR signal strength influence fate by inducing Id3, an antagonist of E protein DNA binding. While E proteins clearly play a central role, their influence on developmental outcomes is almost certainly modulated by additional transcription factors and the extracellular signals that control their expression and function. Accordingly, these fate decisions are too complex to be understood by focusing on one gene or pathway and so require the comprehensive, network-based approach pioneered by Murre. We will employ this approach to elucidate the E protein targets that are crucial for these fate decisions, as well as, the DNA-binding proteins and signaling cascades with which they cooperate. In doing so, we will exploit the ?? TCR transgenic (Tg) model, KN6, whose known selecting ligand, the non-classical MHC-I molecule T10d, can be manipulated to alter TCR signaling. In Aiml, we will employ KN6 Tg mice, and endogenous T10/22 reactive ?? progenitors, to determine how specific ablation of the T10/22 ligand affects ?? lineage commitment, repertoire selection, and effector function. Aim2 will exploit the network approach described above to determine how Id3 is able to promote development of V?2+ and V?3+ ?? T cells, but restrains the development of V?1.1+ innate ?? T cells. Aim3 will exploit our novel marker of ?? lineage commitment, CD73, to determine whether ?? lineage commitment and specification of effector fate are separable or occur simultaneously. The molecular processes defining lineage commitment will be elucidated through the comprehensive network being assembled in Project 4, and by genetic tests of the resulting molecular model. These efforts require the capabilities of all program members and promise to reveal critical new insights into how ?? T cell development is controlled. RELEVANCE: ?? T cells regulate inflammation, preserve epithelial barriers, and are particularly adept at killing cutaneous tumors. Accordingly, understanding the molecular processes controlling their development and function may enable their manipulation for therapeutic benefit. Moreover, our investigation of molecular effectors controlling T lineage commitment is also of fundamental importance for other developmental processes, since control of cell growth and differentiation is a recurring theme in development and transformation.